Human immunodeficiency virus type 1 (HIV-1) is the etiological agent that causes acquired immunodeficiency syndrome (AIDS). According to the AIDS Epidemic Update (UNAIDS, December 2007) approximately 36 million people are living with human immunodeficiency virus type-1 (HIV-1). Although the most severely affected areas are in Sub-Saharan Africa and South-East Asia, more than 2 million people are living with this disease in North America, Western and Central Europe. A significant increase in HIV infection in African Americans has been reported and HIV/AIDS was the leading cause of death among the African American women in 2002 in the US. Therefore, the AIDS epidemic is still a major health concern worldwide. The clinically useful anti-HIV drugs are primarily targeted to the reverse transcriptase (RT) and protease (PR), two vital enzymes in HIV-1 life cycle; however, a new drug targeted to another essential enzyme, integrase, has been recently approved by the United States Food and Drug Administration. The introduction of highly active anti-retroviral therapy (HAART) has significantly contributed to the decreased morbidity and mortality among HIV-1 infected individuals. However, the development of resistance to those drugs often poses a serious threat to the treatment options available to patients.
After an intense effort for more than 10 years a peptide-based drug targeted to the HIV-1 entry, T-20 (enfuvirtide), was developed and approved by the FDA in early 2003. The drug, although expensive, showed its potential in treating patients who are non-respondent to the other available drugs. This success is the testament to the fact that it is important to identify critical steps in HIV-1 life cycle and use them as new targets for possible intervention of HIV-1 infection. Recent reports of failure of HIV vaccine trials and microbicide trials validated the critical need to identify and utilize newer targets to develop new classes of anti-HIV-1 therapies.
The HIV-1 genome is composed of three major genes, gag, pol and env. The gag gene encodes the Gag polyprotein, the critical structural protein of HIV-1, whereas pol encodes viral enzymes, such as reverse transcriptase (RT), protease (PR) and integrase (IN), essential for HIV life cycle and env encodes the viral envelope proteins (Env). Assembly, a critical step in the HIV-1 life cycle, is generally thought to occur through the controlled polymerization of the Gag polyprotein, which is transported to the plasma membrane, where assembly takes place. Virus particles are then formed and bud out as spherical immature non-infectious particles. Immediately after budding, the particles undergo a process known as maturation. During this step, the Gag protein is sequentially cleaved by viral protease to matrix (MA), capsid (CA), nucleocapsid (NC), and p6 domains, as well as two spacer proteins, SP1 and SP2. This process triggers a dramatic change in the morphology of the particles, and an electrodense core is formed surrounded by the conical capsid. The formation of the mature capsid plays a critical role in viral infectivity. Gag has been shown to be essential and sufficient to form virus-like particles (VLP) in the absence of any other proteins or viral RNA. This led to many subsequent studies in determining the regions of gag responsible for HIV-1 assembly by genetic approaches. Data obtained through deletion, insertion and substitution of amino acids in Gag have identified three regions of Gag most important for viral assembly. They have been termed as the membrane binding domain or M-domain, the interacting domain or I-domain and the late domain of L-domain.
The immediate post entry events after the fusion of the infected virions are not clearly understood. However, it is clear that uncoating and disassembly of the mature viral core to release viral genetic material for further processing is critical for the HIV-1 life cycle. A number of studies involving Gag mutations have indicated that Gag may play a critical role in these early events in HIV-1 life cycle.
During HIV-1 assembly and morphogenesis, Gag organizes into two completely different arrangements, immature and mature forms. In case of immature form, Gag remains intact, whereas the mature form is composed of proteins cleaved by viral protease. The formation of this mature particle is essential for HIV-1 infectivity and the capsid protein obtained from the Gag cleavage product plays central role in forming the conical core of the virus that surrounds the viral genome. The capsid protein (CA, p24) is a hydrophobic protein consists of two domains, N-terminal domain (NTD, amino acids 1-145) and C-terminal domain (CTD, amino acids 146-231). These two domains are connected with a 5-amino acid linker and fold independently of each other. Although the exact nature of the CA-CA contacts and their interactions in immature particles are not fully known, in mature particles, the CA lattice has been modeled based on the structural studies and the image reconstruction by cryo-electron microscopy of pure mature virions and assembled virus-like particles. HIV-1 capsid plays crucial role in viral assembly, maturation and early post-entry steps. Mutations of the capsid in both NTD and CTD have been shown to lead to defects in viral assembly and release. In addition, the capsid has been shown as a dominant determinant of retrovirus infectivity in non-dividing cells.
The NTD of the HIV-1 capsid binds to cyclophylin A and is important for viral core formation; however, critical determinants of Gag oligomerization, essential for viral assembly and maturation, are located in the C-terminal domain of capsid. In addition, the CTD encompasses the most conserved segment of Gag known as the major homology region (MHR). Mutation of this conserved region causes severe defects in viral assembly and maturation. The isolated CTD of HIV-1 capsid forms a dimer in solution with the same affinity as the full-length capsid. It has been shown that CTD dimerization is the major driving force in Gag assembly, virus budding and maturation. Several structures of the CTD dimer have been reported, which provided critical information on the dimer interface. Mutation of the interface residues in the CTD monomer disrupts the dimer formation, impair capsid assembly and maturation and renders virus non-infectious.
Taken together, it is evident that capsid plays an important role in HIV-1 assembly and maturation and has been recognized as a potential target for developing new generation of drugs for AIDS therapy.
Protein-protein interactions play a key role in a range of biological processes such as antigen-antibody interaction, viral assembly, programmed cell death, cell differentiation and signal transduction. Therefore, controlling these interactions offers opportunities for developing novel therapeutic agents. However, inhibiting these processes by traditional drug discovery techniques may be complicated and challenging due to the shallow binding interfaces and relatively large interfacial areas involved in most protein-protein interactions. Until recently, it was believed to be virtually impossible to inhibit protein-protein interactions. However, this notion is now changing due to recent advances in this area. In addition, recent studies on crystallized antigen-antibody complexes have shown that only a limited number of residues from each protein partner are involved in mediating protein-protein interactions. These restricted areas at the binding interfaces are known as ‘hot spots’, small areas of bumps and holes that account for most of the protein interface's free energy of binding. Therefore it has been established that inhibitory molecules do not have to cover the entire binding interface to inhibit protein-protein interactions and that targeting these ‘hot spots’ may potently inhibit interprotein contacts.
Dimeric proteins provide a classical example of protein-protein interactions through surface recognition. There are several examples of competitive inhibitors of protein dimerization that exploited the structure of the protein interfaces. For example, interfacial peptides have been shown to inhibit dimerizaton of HIV-1 integrase, protease and reverse transcriptase. However, none of these peptides is clinically useful due to their lack of cell permeability.